Journal of Chemical Ecology https://doi.org/10.1007/s10886-019-01085-1

Plant-Based Natural Product Chemistry for Integrated Pest Management of Drosophila suzukii

Ian W. Keesey1 & Nanji Jiang2 & Jerrit Weißflog3 & Robert Winz4 & Aleš Svatoš3 & Chen-Zhu Wang2 & Bill S. Hansson1 & Markus Knaden1

Received: 17 April 2019 /Revised: 11 June 2019 /Accepted: 14 June 2019 # The Author(s) 2019

Abstract Since the first reports of damage by Drosophila suzukii, the spotted-wing Drosophila (SWD), over a decade ago in Europe, widespread efforts have been made to understand both the ecology and the evolution of this insect pest, especially due to its phylogenetic proximity to one of the original model organisms, D. melanogaster. In addition, researchers have sought to find economically viable solutions for the monitoring and management of this agricultural pest, which has now swept across much of Europe, North America and Asia. In a new direction of study, we present an investigation of plant-based chemistry, where we search for natural compounds that are structurally similar to known olfactory cues from parasitoid wasps that in turn are well- described ovipositional avoidance cues for many Drosophila species. Here we test 11 plant species across two plant genera, Nepeta and Actinidia, and while we find iridoid compounds in both, only those odorants from Actinidia are noted to be detected by the insect antenna, and in addition, found to be behaviorally active. Moreover, the Actinidia extracts resulted in oviposition avoidance when they were added to fruit samples in the laboratory. Thus we propose the possible efficacy of these plants or their extracted chemistry as a novel means for establishing a cost-effective integrated pest management strategy towards the control of this pest fly.

Keywords Olfaction . Chemical ecology . Spotted wing Drosophila . Insect behavior . Parasitoid . Oviposition . IPM

Introduction insect has been unrelenting in its invasion and its spread of economic damage within not just Europe (Tait et al. 2018), but Since its identification in Spain and Italy in 2008 (Cini et al. also across North America and Asia (Cloonan et al. 2018). 2012), Drosophila suzukii, the spotted wing Drosophila Since then, a variety of publications have increased our (SWD), has continued to spread and remain a consistent prob- knowledge regarding its chemical ecology (Adrion et al. lem throughout Europe for agricultural and commercial busi- 2014; Crava et al. 2016;Hwangetal.2005; Karageorgi nesses that produce a wide variety of berry fruits or their et al. 2017; Keesey et al. 2015; Ramasamy et al. 2016). associated products. Eleven years later, as of 2019, this pest However, even given the prevalence of D. suzukii as a target

Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10886-019-01085-1) contains supplementary material, which is available to authorized users.

* Ian W. Keesey 2 State Key Laboratory of Integrated Management of Pest Insects and [email protected] Rodents, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, Beijing 100101, China * Bill S. Hansson [email protected] 3 Mass Spectrometry/Proteomics Research Group, Max Planck * Markus Knaden Institute for Chemical Ecology, Hans-Knöll-Straße 8, [email protected] D-07745 Jena, Germany

4 The New Zealand Institute for Plant & Food Research, 120 Mt. 1 Department of Evolutionary Neuroethology, Max Planck Institute for Albert Road, Mt. Albert, Auckland 1025, Private Bag 92169, Chemical Ecology, Hans-Knöll-Straße 8, D-07745 Jena, Germany Auckland 1142, New Zealand JChemEcol for research during the last decade, there is still a large gap in for at least some parasitoid species within the Leptopilina applicable technology for monitoring and control of this insect genus, the same compounds seem to also be used as marking pest. pheromones that help to avoid competition for Drosophilid Commercial synthetic production of compounds known to hosts (Pfeiffer et al. 2018). However, again, the commercial be repellent towards the Drosophila genus, such as geosmin synthetic production of these known repellent wasp com- (Stensmyr et al. 2012), are prohibitively expensive to produce pounds is both expensive and labor-intensive, as would be for large-scale usage in the field (Cloonan et al. 2018; the collection of these odors from the mass production of Wallingford et al. 2016a, b), and their application on the fruit parasitoid wasps, which themselves are much smaller than a shortly before harvest (i.e. when D. suzukii mainly infests the single D. melanogaster adult (Ebrahim et al. 2015; Stökl et al. berries) might have a negative impact on fruit quality 2012;). Therefore, collection of these odorants from these tiny (Diepenbrock et al. 2017; Leach et al. 2018). Forinsect pests, Hymenopterans may prove to be too inefficient for large-scale natural-product chemistry, including both microbial-derived production. and plant-derived sources have been shown to be efficient As iridoid compounds that bear strikingly similar structural (Abdel-Sattar et al. 2010; Maia and Moore 2011;Pavela chemistry towards the wasp pheromones have also been de- 2016). For example, the leaves of pepper trees and their ex- scribed in plants like catnip, e.g. Nepeta Lichman et al. 2018; tracts have been used to repel house flies (Musca domestica) Sherden et al. 2018; Shim et al. 2000;), and kiwifruit, e.g. in Ethiopia (Abdel-Sattar et al. 2010), and peppermint oils Actinidia (Lu et al. 2007;Matichetal.2003; Tatsuka et al. have been shown to be repellent towards D. suzukii in a lab- 1990; Twidle et al. 2017), we therefore hypothesized that oratory setting (Renkema et al. 2016). However additional, these plant genera may provide a natural source of functional economically viable strategies to combat D. suzukii still need odorants to repel D. suzukii adults. Commercial products al- to be identified, as most research has focused on attractive ready exist from Nepeta plants, such as dried Nepeta leaves, as odors for monitoring or bait and kill strategies for population well as extracted oils or synthetic compounds that have similar control (Klick et al. 2019; Landolt et al. 2012;). bioactivity to those identified from Nepeta varieties, where all An area of rapidly growing research has been the study of products are sold as cat toys or feline attractants (Bol et al. biocontrol options for D. suzukii using either existing, native 2017). Catnip oil extracts have also been shown previously to parasitoid wasps (e.g. those found in Europe) or other parasit- possess some behavioral repellency against other Dipterans, oids from evolutionary associations within the original habi- such as the stable fly, Stomoxys calcitrans, and other biting tats occupied by D. suzukii (e.g. those parasitoids from China, flies, which are pests of livestock (Zhu 2011). However, to our Korea and Japan). Here, although numerous parasitoid wasp knowledge, no integrated pest management (IPM) strategy species have been examined, there appear to be barriers to the has ever been examined utilizing kiwifruit plant materials. effectiveness of these biocontrol agents (Asplen et al. 2015; In order to test our hypotheses that natural, plant-based Daane et al. 2016), especially due to variation in the immune chemistry may provide possible IPM solutions towards system of D. suzukii and its superior ability to encapsulate D. suzukii, we therefore searched for parasitoid-like iridoid wasp eggs relative to D. melanogaster adults (Chabert et al. compounds in the plant extracts from seven total Nepeta plant 2012; Kacsoh and Schlenke 2012). However, there may still species as well as from four total Actinidia species. In addi- be future success in this tactic, once a suitable species of tion, we tested whether any of these plant-produced odorants parasitoid wasp has been uncovered to more effectively man- could activate the parasitoid-specific olfactory sensory neuron age these flies in natural and agricultural ecosystems. In asso- pathway in the adult fly. Lastly, we sought to examine whether ciation with parasitoids, a study that recently focused on these plant-based odorants were sufficient to induce oviposi- D. melanogaster identified three pheromone components of tional avoidance by D. suzukii adults in the laboratory. the Drosophila-specific parasitoid, Leptopilina boulardi (Hymenoptera: Figitidae: Eucoilinae), where each of the three pheromone components (i.e. iridomyrmecin, nepetalactol, and Methods and Materials actinidine) from the wasp body wash was shown to activate the same parasitoid-specific olfactory sensory neuron (OSN) Plant Sample Extraction Fresh samples of 8 varieties across in D. melanogaster. Moreover, it was shown that this neural 7 species within the Nepeta genus were collected from activation pathway in turn leads to oviposition avoidance of potted plants grown outdoors, including leaves as well as these parasitoid odorants by the adult fly (Ebrahim et al. inflorescences, which were each collected separately. Plant 2015). Importantly, this study also examined behaviors related extracts were generated using 1 hr washes of cut plant to other flies within the genus, including D. suzukii, where the material (2–3 g) in hexane or separately via washes in presence of these three wasp pheromones also generated sig- methanol, and collections were aided by periodic mixing nificant avoidance, both in larvae and adults. Interestingly, (vortex genie 2; www.scientificindustries.com). Extraction although being shown to act as sex pheromone in the wasp, protocols for plant materials followed established methods JChemEcol and solvents for iridoid compounds as well as from the well as for actinidine and nepetalactol (Beckett et al. 2010) literature related to these plant genera (Hallahan et al. using (R)-citronellal as starting material. Dilutions of each of 1998;Luetal.2007; Tatsuka et al. 1990;). Liquid extrac- the synthetic odors were prepared in hexane for both electro- tions were filtered to remove any loose particulates, and physiological and behavioral trials, and synthetic chemicals then stored at −20 °C until ready for GC-MS and insect were stored with nitrogen in sealed containers and kept in trials. Fresh samples of flowers and leaves from 4 plant the dark at -80 °C when not in use. species and multiple genotypes within the Actinidia genus were also collected and then shipped from New Zealand to Fly Stocks Wildtype D. suzukii (14023–0311.01) were ob- Germany during the northern hemisphere summer 2018 tained from the former University of California San Diego (winter in New Zealand). The samples were collected via Drosophila Stock Center, which is now the National the Plant & Food Research team in Auckland. These sam- Drosophila Species Stock Center (Cornell University; ples were kept frozen in temperature monitored containers http://blogs.cornell.edu/drosophila/). All experiments with during transport (www.WorldCourier.com), with alarms set wildtype D. melanogaster were carried out with the to trigger if temperatures reached above −15 °C, which Hansson Canton-S (CS) laboratory strain, which were orig- occurred only once (just following arrival in Germany). inally obtained in 2008 from the Bloomington Drosophila Frozen Actinidia samples were then immediately stored Stock Center (www.flystocks.bio. indiana.edu). Fly stocks again at −80 °C until ready for processing in the were maintained according to previous studies (Keesey laboratory. Tissue samples were broken off from frozen et al. 2015, 2019), and for all behavioral experiments we Actinidia materials (leaves and flowers; 2–3g)andplant used 4 to 7 day-old flies. extracts were collected using 1 hr washes in hexane and methanol solvent as previously described for Nepeta plant Single-Sensillum Recordings (SSR) In order to assess olfacto- tissues. ry similarities and differences between these two Drosophilia species, we conducted single-sensillum re- Gas Chromatography Mass Spectrometry (GC-MS) Chemical cordings of both flies. Adults were held immobile within analyses were performed on all plant extract collections as plastic pipette tips, with only the head of the fly exposed, described previously (Keesey et al. 2015, 2017;Qiaoetal. where the third antennal segment (funiculus) and com- 2019), where plant materials were generated via external pound eye were both stabilized against a glass cover slip washes (as opposed to plant materials being ground, crushed to aid tungsten electrode penetration. Sensillum type was or homogenized). The NIST mass-spectral library identifica- identified using established diagnostic odors and via tions were confirmed with chemical standards where possible, searching established antennal regions of highest density including the high-purity synthetic isomers of the parasitoid probability (Ebrahim et al. 2015; Lin and Potter 2015). A pheromones from Leptopilina boulardi wasps, which were reference electrode (tungsten) was inserted into the com- previously identified and published from our Institute pound eye closest to the targeted antenna, while a recording (Ebrahim et al. 2015). Plant extracts for all Actinidia and electrode (tungsten) was used to pierce individual sensillum Nepeta species and tissue types were prepared in both hexane types along the antenna for potential screening and identi- and methanol solvents (to provide additional diversity across fication using the appropriate diagnostic odors. Odor stim- polarity, as well as to maximize chemical identification and ulus preparation and delivery for SSR experiments follow- differentiation of each compound within these plant samples). ed previously established procedures (Keesey et al. 2017). Moreover, all plant samples were run across both HP5 and HP-INNOWax GC columns during our chemical analyses. Oviposition Assays As a means to examine the role these Raw data files (Agilent ChemStation) are available with the iridoids play in the ecology of D. suzukii,wesoughttotest online version of this publication. Chemical standards were egg-laying decisions in the presence of these odors. generated from in-house laboratory sources (Mass Experiments were carried out in large mesh cages (50 cm × Spectrometry Research Group, Max Planck Institute for 50 cm × 50 cm; https://shop.bugdorm.com/bugdorm-4f4545- Chemical Ecology) and were of the highest purity possible. insect-rearing-cage-p-31.html, BugDorm-44,545 F) which In total 6 chemical standards were produced, including: were placed inside walk-in growth chambers (12 hr Light: (4R,4aR,7R,7aS)-(−)-iridomyrmecin (87% pure), a 1:1 mix Dark, 70% humidity, 23 °C). Oviposition choice plates were of (4S ,4aR ,7R ,7aS )-(+)-isoiridomyrmecin and generated with standard Drosophila diet, with a single freshly (4R,4aR,7R,7aS)-(−)-iridomyrmecin, (R)-actinidine (95% smashed blueberry in the center, and each blueberry had either pure), (R)-actinidine (20% EtOAc) and (1S,4aR,7R,7aS)- 50 μl solvent control (hexane) or 50 μl of treatment (also in nepetalactol (Supplementary Figure 2). Syntheses were con- hexane) added over it (similar to previous oviposition ducted as described previously for stereoisomers of methods described for D. suzukii experiments (Karageorgi iridomyrmecin (Stökl et al. 2012;Fischmanetal.2013)as et al. 2017)). However, we had several difficulties initially JChemEcol with establishing oviposition with D. suzukii,thuswemade we utilized red paper cones as the trap entrance. This has been several modifications. First, each cage had moistened white shown to enhance capture for larger Drosophilids like tissue paper in the center. This reduced desiccation pressures D. suzukii (as opposed to smaller pipette tip entrances), as well over the behavioral trial by providing a constant source of as shown to enhance capture for those fly species that are water, it provided a surface texture that reduced turtling of perhaps more visually driven during search or host navigation flies (e.g. stuck on their backs), as opposed to the slippery paradigms (Keesey et al. 2019). One trap contained a dam- plastic bottom surface of the cages, and the tissue paper also aged blueberry with 50 μl hexane solvent (control), while the generated a reproducible distance between control and treat- other trap contained a damaged blueberry and 50 μloftreat- ment plates (20 cm). This strategy boosted survivorship of ment diluted in hexane. Both of the traps had 100 μlof adult flies to over 90% during these behavioral trials. laboratory-grade mineral oil added to assist in killing flies Second, our packaged blueberries varied considerably in size inside after they made a choice during the 24 hr experiments. and color, thus control and treatment blueberries were selected Trials were conducted as noted before (12 hr Light:Dark cy- to match each other in size, weight and color. Lastly, despite cle, 70% humidity, and 23 °C), with ten total replicates. The our best efforts to utilize agar plates, or transparent media to attraction index was calculated as AI = (T-C)/20 where a total aid in egg counting, we could not get sufficient oviposition of 20 flies were allowed to make a choice between the treat- from D. suzukii females using these media types. Therefore, ment (T) and control (C) trap containers. we chose to use the same standard diet that the flies are already reared upon, which we first transferred to oviposition plates Statistical Analyses and Figure Generation Statistical analyses and spread evenly, then provided texture, and finally placed were conducted using GraphPad InStat 3 (https://www. our freshly smashed blueberry in the center (Fig. 5d). Each graphpad.com/scientific-software/instat/), while figures were cage had a total of 20 females and 10 males released inside to organized and prepared using R Studio, Microsoft Excel and ensure optimal mating status (where flies were selected after Adobe Illustrator CS5. Normally distributed data were

2–4 min of anesthesia by cooling at −20 °C; no CO2 was used analyzed using two-tailed, paired t-tests or a one-way analyses to prepare the flies for oviposition trials). Appropriate age and of variance (ANOVA; Tukey-Kramer multiple comparison reproductively viable females were selected as those which test), where means with the same letter are not significantly released and displayed a single egg from their ovipositor dur- different from one another. Boxplots represent the median ing anesthesia. After transferring adults to the cages, flies were (bold black line), quartiles (boxes), as well as the confidence given 48 hr to adjust to the enclosures as well as to lay eggs, intervals (whiskers). Color-filled boxplots denote significance and then both the control and treatment plates were removed from zero, while empty (white) boxes were not significantly for counting and subsequent photo analyses. Eggs that different from zero. Error bars presented for bar graphs repre- hatched prior to plate removal were still countable, as the sent the standard deviation. chorion (shell) was still visible after larval emergence, and although most intact eggs were deeply buried by the females in our substrate, the pairs of breathing tubes were still visible Results to facilitate accurate egg counts. We also found it useful to use a black and white camera, as opposed to full color, in order to Plant Chemistry We first sought to examine whether the plant better discern eggs that had been completely buried (Fig. extracts of any of the tested Nepeta or Actinidia species would 5d, e). The oviposition index (OI) was calculated as OI = (T- indeed include the parasitoid compounds that are known to C)/(T + C), where T is the number of eggs on the treatment induce oviposition avoidance in Drosophilid flies (Ebrahim plate, and C is the number of eggs on the control plate. We also et al. 2015) (GC-MS; Fig. 1). For the seven Nepeta plant counted total eggs per cage across both the control and treat- species, we identified large amounts of two as-of-yet uniden- ment to examine consistent female fecundity when generating tified isomers of nepetalactone in the leaves (but not in the the oviposition indices. However, in trials with the synthetic flowers). However, we did not find any of the previously parasitoid odor mixture as our treatment, we note a significant identified parasitoid odors (i.e. iridomyrmecin, nepetalactol, decrease in the total observed fecundity or egg deposition, or actinidine) within these plant samples (Figs. 1, 3; although all plant tissue experiments had nearly identical total Supplementary Figure 1). When we next examined the ex- egg numbers across the eight replicates (Fig. 5b). tracts of the four different Actinidia species, we found at least traces and in some species even large amounts of several Trap Assays As we had established a behavioral effect of these iridoids that structurally resembled all three previously de- iridoids on egg laying, we next wanted to examine attraction scribed parasitoid compounds (Figs. 2, 3). Here the consistent- and aversion using these odors. Experiments were performed ly highest amounts of these three compounds were present in in smaller plastic enclosures (10 cm × 10 cm × 8 cm), which the leaf extracts of A. polygama. Also flower extracts of this contained two trap containers. To assist in total capture rates, species contained nepetalactol and some traces of JChemEcol

1 Nepeta faassenii (leaf) Plant Extract 2 123.1 81.1 166.1 O ab95.1 nepetalactone (1)

O 69.1

109.1

41.1 nepetalactone 55.1 Intensity

138.1 29.1 151.1

m/z (R) - nepetalactol (10-5)

123.1 166.1

81.1 nepetalactone (2) Intensity (TIC) 95.1 (R) - actinidine (10-5) 69.1 109.1 41.1

Intensity 55.1

29.1 137.1 (-) iridomyrmecin (10-5) 151.1

8.00 11.00 Retention Time (minutes) m/z Fig. 1 Gas chromatography mass spectrometry (GC-MS) analyses of There was no retention time or mass spectral overlap between Nepeta Nepeta genus plants. a Shown at top is the leaf extract collected with plant extracts and synthetic parasitoid compounds; however, Nepeta hexane, and below are the three synthetic standards of the parasitoid plants did contain high amounts of two unidentified isomers of pheromones for Leptopilina boulardi wasps, each odor of which has been nepetalactone. In total we tested 7 different species within this genus of shown to be aversive in several Drosophila species within the subgenus plant, but did not find any matches for the parasitoid odors. b Mass Sophophora, including D. suzukii adults and larvae (Ebrahim et al. 2015). spectra for the two main isomers of nepetalactone found in these plants iridomyrmecin (Fig. 3). We confirmed the presence, absence adults. We next tested whether any of the plant compounds and identity of these plant-produced iridoid compounds by would activate these same olfactory sensory neurons that are comparing the retention times as well as the mass spectral known to govern oviposition avoidance towards Leptopilina signature for each chromatogram peak to synthetic parasitoid parasitoids (Fig. 4b). Here we tested the response of potential reference standards (Figs. 1, 2a, b), where there was a close to ab10 sensilla in D. suzukii towards both the diagnostic odor perfect match for both retention time and mass spectral EI-MS (actinidine, diluted to 10−5 in hexane) as well as hexane ex- data between the synthetic odorants and the plant-derived tracts from our Actinidia plant tissues (Fig. 4b). In the smaller iridoid compounds from Actinidia species but not from any “B” neuron of this sensillum type (which co-expresses Or49a of the Nepeta varieties (Figs. 1, 2a, b). and Or85f in D. melanogaster), we found strong responses to both the synthetic reference compound, actinidine, as well as Electrophysiology of Parasitoid-Detecting Sensilla Using a to the leaf extracts of A. polygama and A. macrosperma (Fig. synthetic parasitoid pheromone compound, Actinidine, whose 4b, c). However, none of the extracts from Nepeta plants re- detection by the fly is highly specific and that activates only a sulted in any activation of the neuron of interest (Fig. 4c), single olfactory sensory neuron type (i.e. ab10B in suggesting that the Nepeta-specific iridoids do not function D. melanogaster), we were able to map the locations in as ligands for this neuron. The larger “A” neuron response D. melanogaster as well as in D. suzukii of the antennal profile for the ab10 sensillum of D. suzukii is also less sensi- basiconic sensillum “ab10-like” types (Fig. 4a). This exami- tive in its response to benzyl butyrate, which is currently the nation corresponded to and confirmed previous work on this best ligand for D. melanogaster flies. This neuron may thus be receptor and sensillum type for D. melanogaster (Ebrahim slightly different in its ligand spectra or odorant tuning com- et al. 2015). The overall body and antennal size of D. suzukii pare to D. melanogaster.However,the“B” neuron showed is significantly larger than D. melanogaster, as was shown nearly identical ligand spectra and odor sensitivity between recently (Keesey et al. 2019), thus it was not surprising that the two tested Drosophila species. Here the synthetic parasit- we noted more potential ab10 sensillum numbers for this pest oid pheromones (e.g. actinidine) proved to be the best diag- species, simply because it has a higher total number of sensilla nostic odor for identifying ab10 sensillum types throughout and more total basiconic sensilla than in D. melanogaster the antenna of both fly species. Therefore, we conclude that JChemEcol

Actinidia polygama (leaf) 2 Synthetic Plant Extract 1 ( R ) - nepetalactol ab135.1 135.1 168.0 168.0 81.1 41.1 97.1 97.1 41.1 81.1 108.1 3 108.1 Intensity

HO

132.1 132.1 ( R ) - actinidine O

-5 (R) - nepetalactol (10 ) 147.1 147.1

117.1 117.1 Intensity

77.1 77.1 Intensity (TIC) N -5 (R) - actinidine (10 ) (-) iridomyrmecin 95.1 95.1 81.1 81.1 67.1 67.1

O 109.1 109.1

O 41.1 41.1 55.1 55.1 (-) iridomyrmecin (10-5) Intensity

8.00 11.00 m/z m/z Retention Time (minutes) Fig. 2 Gas chromatography mass spectrometry (GC-MS) analyses of and flowers from both male and female plants. Each tested sample Actinidia genus plants. a Shown at top is the leaf extract collected with contained potentially bioactive compounds, although those from hexane, and below are the three synthetic standards of the parasitoid A. polygama consistently contained the largest amounts. b Mass spectra pheromones for Leptopilina boulardi wasps, each odor of which has for each of the three plant odors of interest and the three synthetic been shown to be aversive in several Drosophila species within the parasitoid compounds, where retention time and mass-spectral signature subgenus Sophophora, including D. suzukii adults and larvae (Ebrahim are nearly identical for all three odorants et al. 2015). In total we examined 4 species of Actinidia, including leaves the ab10B neuron appears to be highly conserved across the odors. Thus, this neural circuit, both for detection via the an- Drosophila genus, as has been suggested previously, and it is tenna and for avoidance behavior, still appears to be narrowly narrowly tuned specifically towards these iridoid compounds tuned in all Drosophila species tested thus far. In addition to (Ebrahim et al. 2015). oviposition trials, we also examined the attraction index of adult D. suzukii towards the parasitoid odors and the plant Behavioral Aversion with Plant Extracts and Parasitoid Odors extracts (Fig. 5c). Here, similar to the previously described Having shown that the Actinidia extracts contain parasitoid- behavior for Drosophila adults towards the body washes of like compounds via our GC-MS analyses, and subsequently, L. boulardi (Ebrahim et al. 2015), we did not observe any that these plant extracts can activate the fly olfactory circuit significant behavioral aversion towards the parasitoid mix that is dedicated towards the avoidance of parasitoids, we next nor any of the plant extracts. This suggests that adult flies examined whether the plant extracts are sufficient to induce show only oviposition avoidance, and perhaps not any long- oviposition avoidance in D. suzukii. As shown previously range repellency or feeding aversion towards these chemical (Ebrahim et al. 2015), when adult D. melanogaster flies were stimuli. Future research will need to address either greenhouse given the choice between oviposition plates with solvent con- or field-based testing of the efficacy of Actinidia plant extracts trol or plates with a mix of synthetic parasitoid pheromone to combat D. suzukii oviposition. odors, the adult females significantly preferred to lay eggs on the control, and avoided egg laying near the parasitoid odors (Fig. 5a). We found a similarly significant avoidance Discussion for oviposition when utilizing our new plant extracts taken from A. polygama and A. macrosperma (where D. suzukii The world-wide pest, Drosophila suzukii, oviposits in high females again preferred the solvent control over the treat- value crops like cherries and soft fruits, including many ment). However, no significant behavioral aversion was found kinds of berries and grapes. As this insect infests fruits just for any of the three tested Nepeta plant extracts (Fig. 5a), even before harvest, any pesticide treatment is difficult to utilize though these plants contained two isomers of nepetalactone, due to the pesticide-specific pre-harvest intervals. We which are chemically similar in structure to the parasitoid therefore investigated whether we could identify known JChemEcol

Leaves Flowers

Species (Variety) NepetalactoneIridomyrmecinActinidine Nepetalactol NepetalactoneIridomyrmecinActinidine Nepetalactol

Nepeta faassenii (dropmore) +++ ______

Nepeta faassenii (gletschereis) + ______

Nepeta racemosa (snowflake) +++ ______

Nepeta sibirica + ______

Catnip Nepeta cataria (citridora) + ______

Nepeta pratti + ______

Nepeta subsessilis (sweet dreams) + ______

Nepeta grandiflora (dawn to dusk) + ______

_ _ Actinidia arguta trace trace ++ trace _ +

_ _ Actinidia valvata trace +++ trace _ + _ _ _ Actinidia macrosperma trace +++ + trace ++ Kiwifruit _ _ _ Actinidia polygama +++++ +++ trace +++ Fig. 3 List of all tested plant extracts (leaves and flowers) for Nepeta and as compared to flowers. One species, Actinidia polygama, consistently Actinidia species. Names of each species and variety that were tested, produced the largest amount of our compounds of interest, and represents across both catnip (Nepeta) and kiwifruit (Actinidia) genera. While the best target for additional study of these iridoid odors, including the catnip varieties produced large amounts of unidentified isomers of natural product that is the namesake of this genus of plant, actinidine. nepetalactone, they did not generate any isomers of the behaviorally (− = odor was not detected by GC-MS; trace = threshold levels detected; active compounds of interest. However, each species and genotype of + = low amounts detected; ++ = moderate amounts; ++++ = highest kiwifruit produced one or several isomers of the parasitoid odors; amounts of major iridoid components found) moreover, leaf materials always contained larger amounts of the odors

oviposition deterrents for D. suzukii in natural, plant-based also been described from the genome and from gene extrac- extracts that could in turn be customized towards novel tions of D. suzukii antennae (Ometto et al. 2013;Adrion IPM strategies while still presumably safe for human con- et al. 2014; Ramasamy et al. 2016). Interestingly, sumption. Like many other Drosophilid flies, D. suzukii D. suzukii also shows a potential gene duplication of detects and avoids the pheromone compounds of the para- Or49a (as compared to D. melanogaster, which only has a sitoid wasp Leptopilina boulardi (Ebrahim et al. 2015). The single copy), and this may in the future offer some behav- highly specific olfactory circuit for this ovipositional avoid- ioral significance in regard to studies of avoidance for other ance has been described in detail for a close relative, parasitoids or parasitoid odors (Ramasamy et al. 2016). For D. melanogaster, where two olfactory receptors (OR49a example, D. suzukii may show stronger aversion towards an and OR85f, which are co-expressed in one olfactory senso- as-of-yet-undescribed parasitoid species, or perhaps to- ry neuron type, the ab10B olfactory sensory neuron (OSN) wards a native parasitoid (i.e. Leptopilina japonica), as can detect the wasp-specific iridoid compounds, namely provided by a novel odorant tuning via this receptor dupli- (−)-iridomyrmecin, (R)-actinidine, and (S)-nepetalactol. cation. In D. melanogaster the activation of the ab10B neu- While activation of Or49a seems to be highly specific to- ron was demonstrated to be sufficient to govern oviposition wards the parasitoid-specificisomerofiridomyrmecin, avoidance that the flies exhibit in the presence of live par- Or85f could be activated by several isomers of actinidine asitoid wasps (Ebrahim et al. 2015;Stökletal.2012). We and nepetalactol in previous studies (Ebrahim et al. 2015). therefore hypothesize that these similar compounds identi- Both of these olfactory receptors (Or49a and Or85f) have fied from Actidinia plants, which activated the same JChemEcol

Tungsten Electrode ab D. melanogaster D. suzukii antennal basiconic sensillum (ab10) Or67a Stimulus (0.5 seconds) Or49a/Or85f

Actinidine (10-5) ab10 c 140 b 120 b b 100 Actinidia polygama (Plant Extract; Leaf) 80 60 n = 10 40 Spikes per Second Spikes 20 a a a a 0 Solvent Control (Hexane) -20 -5 )

N. faassenii A. polygama N. grandiflora N. racemosa Solvent ControlActinidine (10 A. macrosperma

Fig. 4 Single-sensillum recordings (SSR) from parasitoid-detecting responses in the same neuron, which has been shown to house Or49a olfactory receptors. a Schematic of D. melanogaster and D. suzukii (3rd and Or85f in D. melanogaster adults. c Quantified SSR responses antennal segment; funiculus) depicting locations of parasitoid-sensitive towards plant extracts and the synthetic parasitoid odors, where only sensillum types. b Shown are single sensillum recordings (SSR) of the tested Actinidia plant species produced activation of this neuron. antennal basiconic sensillum (ab10) responses towards the diagnostic These plant extracts were not significantly different in the strength of parasitoid odor, actinidine, and towards the leaf extract of the plant response from the synthetic odor (actinidine) in spikes per second, but species Actinidia polygama,aswellastowardsthesolventcontrol, the synthetic odor often produced a longer duration of activation than the where each odor was tested across the same sensillum. Both the plant samples. Means with the same letter are not significantly different synthetic parasitoid odor and the extracted plant sample evoke similar from one another (α =0.05) olfactory circuit, could potentiallybeappliedinthefieldto oviposition choices of D. suzukii adults (Fig. 5a, b). It should repel D. suzukii flies from depositing eggs in fruit shortly be noted that we found stronger behavioral avoidance associ- before the harvest period. ated with the synthetic parasitoid odors than the Actinidia Although we tested several plant species of the genera plant samples (Fig. 5a). This may be due to quantitative or Nepeta and Actinidia, which are two genera that are known qualitative differences between the plant extracts and the pure, for their production of iridoid compounds, and while we iden- isolated parasitoid odors, though future work is still needed to tified a variety of iridoids from both plant genera, we found ascertain the rationale for differences that were observed. that only odorants from Actinidia were chemically similar (or Similarly, additional work would be needed to examine any potentially identical) to those previously identified from the effect of these plant extracts on adult feeding or larval parasitoid wasp, L. boulardi (Fig. 2). We furthermore found behavior. that only the Actinidia compounds and plant extracts were Additional work is still needed to confirm the olfactory capable of activating the parasitoid-specific OSNs on the an- receptor (OR) identity of those receptors in D. suzukii adults tennae of both D. melanogaster and D. suzukii flies during that respond to these parasitoid and plant-based compounds. single-sensillum recordings (Fig. 4b). Moreover, that the However, the identity of the olfactory receptors would not iridoids from Nepeta plants were too chemically dissimilar change the viability of plant extracts as a potential IPM tech- from the original parasitoid compounds to serve as ligands nique towards reduction of oviposition in the field by this pest for the wasp-specific olfactory receptors of the fly. Thus, not insect, especially given their functional similarity to those pre- surprisingly, when we next tested behavior associated with viously identified as key stimuli from the ab10 OSN type in plant extracts from both Nepeta and Actinidia plants in ovipo- D. melanogaster adults. That being said, the response profile sition assays, we found avoidance only towards the Actinidia and ligand spectra of the ab10B neuron, which expresses the extracts, while the Nepeta extracts did not affect the olfactory receptors detecting both the plant and parasitoid JChemEcol

Parasitoid Mix Actinidia Extract Nepeta Extract a b c n = 8 45 b n = 10 40 b b Treatment Treatment b b 35 30 a 25 20 Solvent Control Solvent Control 15 10 Average # Eggs Average 5 Preference Index (PI) Preference Oviposition Index (OI) 0 -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 Parasitoid A. polygama N. faasseniiN.racemosa A. macrosperma Parasitoid Parasitoid A. polygamaN. faassenii A. polygama N. faasseniiN. racemosa A. macrosperma d ef

Fig. 5 Ovipositional avoidance by Drosophila suzukii of parasitoid and or either genera of plant extracts as compared to the odor cues from the Actinidia plant extracts. a Oviposition index for adult flies that were solvent control, where there is no significant attraction or avoidance presented a choice between standard diet plates with solvent control or shown to any treatment. d Oviposition assay paradigm, using two petri with treatment, where females preferred to lay more eggs on the side dish plates with damaged blueberries, which were separated by a white without parasitoid or Actinidia extracts. Only the two Actinidia plant tissue paper that provided both a source of water and a standard distance extracts produced behavioral aversion while none of the three Nepeta of separation between control and treatment stimuli. e Colorimageswere species generated any significant preference. The mix of three synthetic taken of oviposition sites in order to count egg numbers; however, due to parasitoid odors produced the strongest repellency for oviposition, or may the ability of D. suzukii females to completely bury their eggs deep within have persisted longest in the environment. b Average number of eggs the substrate, it was often difficult to locate and count eggs. f Utilizing a produced in each oviposition choice assay, where there was no black-and-white digital camera which was mounted to the same significant difference in total eggs laid for any of the plant extracts, microscope, we could instead generate monochromatic or greyscale although the parasitoid mix again showed a significant reduction in images, where it was far easier to discern eggs, even those completely total eggs deposited. c Attraction index (trap assays) of adult buried in the substrate, via the identification of the white pairs of Drosophila towards the olfactory cues from the synthetic parasitoid mix breathing tubes (highlighted with arrows). Means with the same letter odors (Fig. 4b, c), was functionally identical between the two acid structure across all species for this insect genus, similar to examined Drosophila species in this study. Moreover, similar- the high-rate of conservation of other aversion-related recep- ly responding olfactory sensory neurons (OSNs) were found tors such as Or56a (which detects geosmin (Stensmyr et al. in several other Drosophila species that were previously ex- 2012)). However, again, additional molecular genetic work is amined (Ebrahim et al. 2015), where all species that detected still needed to test this hypothesis across additional species. the wasp odorants also exhibited oviposition avoidance to- Correspondingly, we believe that the Actinidia extracts could wards these identified parasitoid odors (Ebrahim et al. act as potential oviposition deterrent for other Drosophilid 2015). Thus we believe that the odorant receptors housed in species, but additional work is still needed to test other species the ab10B neuron most likely share a highly conserved amino beyond D. suzukii adults. JChemEcol

While field trials will be needed to test our proposed plant- microorganisms that we seek to combat, the scientific pursuits based IPM strategy, for example with mixed cultivation using related to IPM as well as evolutionary neuroethology will A. polygama, additional aspects also need to be addressed in continue to generate testable hypotheses and potential avenues tandem. For example, it is unclear whether these plant-based for novel approaches to solve agricultural problems through- compounds from Actinidia plants have any behavioral effect out the world. It is a growing concern that the wide-spread use on either parasitoid or predator recruitment (Glinwood et al. of general pesticides such as neonicotinoids, which have been 1999; Zhang et al. 2006), which might provide additional reported as harmful to bees and other pollinators, will only benefits towards the biocontrol of D. suzukii in the field via continue to hinder beneficial plant-insect interactions in natu- increased predation or parasitism rates. Moreover, it is not ral environments (Van der Sluijs et al. 2013; Whitehorn et al. known whether additional pest insects such as aphids, whose 2012). Thus again, we believe the possibility of utilizing aggregation pheromone blend includes isomers of natural-product chemistry, such as bioactive plant extracts or nepetalactone and nepetalactol, may also be increasingly push-pull alley cropping, as opposed to the generation of new attracted in the field by the compounds (Dawson et al. or more powerful pesticides, may afford a more sustainable 1996), thereby creating additional agricultural risks or other and eco-friendly solution to pest insects such as D. suzukii for direct harm to fruit production efforts. It is also unclear wheth- agriculture in the future. er alley-cropping, agroforestry, or other push-pull strategies afforded by establishing Actinidia plants near, around or with- Acknowledgements This research was supported through funding made in fruit production can provide similar oviposition repellency available by the Max Planck Society. Fly stocks obtained from the Bloomington Drosophila Stock Center were used in this study (NIH to that observed in the laboratory. Alternatively, future re- P40OD018537). We express our gratitude to Kerstin Weniger, Silke search we need to address if only chemical extraction and Trautheim and Daniel Veit for their technical support, guidance and ex- application of Actinidia leaf extracts directly onto the crop will pertise at MPI-CE. We thank Philipp Wein for his assistance in develop- provide suitable quantities of avoidance cues for the efficacy ing some behavioral experiments regarding Drosophila suzukii adults. We also thank Adam Matich and Thomas Paterson for discussion of of this IPM strategy to be successful. For example, dose re- unpublished data and for help in sourcing kiwifruit materials in the field. sponse trials need to be conducted to ascertain effective appli- Lastly, we are very thankful for the efforts and assistance provided by cation. The current conditions for growing Actinidia plants are Danny Kessler and his team at the MPICE greenhouse during the order- similar to those for successful viticulture, thus vineyards may ing, growth and maintenance of all live Nepeta plant samples. provide the most appropriate avenue for testing the planting of Author Contributions I.W.K. designed the study, with assistance from kiwifruit plants as a natural deterrent for D. suzukii across N.J., A.S., B.S.H., and M.K. All behavioral trials were performed by regions that already focus on wine production. However, giv- I.W.K. and N.J., as well as all physiological and GC-MS experiments. en that D. suzukii is only an opportunistic pest of grapes, it J.W. and A.S. synthesized and prepared the synthetic parasitoid com- may be more fitting to address alley cropping in berry fruit pounds. R.W. selected, collected and shipped all Actinidia plant materials. A.S., C.Z.W. and B.S.H. provided financial and scientific support. I.W.K. orchards instead. Future experiments in greenhouses will re- wrote the original manuscript and prepared all figures, while all coauthors veal whether the close vicinity of Actinidia plants is sufficient commented on and made adjustments towards the final version of the to reduce crop loss by oviposition of D. suzukii adults. Future paper. analyses with additional plant species such as Indian nettle (Acalypha indica), valerian herbs (Valeriana officinalisi; Funding Information Open access funding provided by Max Planck Society. Nardostachys jatamansi), snapdragon (Catharanthus roseus), yellowbells (Tecoma stans), and honeysuckle (Lonicera Open Access This article is distributed under the terms of the Creative caerulea; Lonicera tatarica), which have all also been sug- Commons Attribution 4.0 International License (http:// gested to produce different kinds of iridoids (Bol et al. 2017); creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give Scaffidi et al. 2016;, might reveal further potent D. suzukii or appropriate credit to the original author(s) and the source, provide a link other Dipteran deterrents from natural or plant sources. It has to the Creative Commons license, and indicate if changes were made. also been shown previously that different isomers of iridoid compounds can have varying degrees of bioactivity (Civjan 2012; Ebrahim et al. 2015; Stökl et al. 2012), thus more work is needed to address stereochemistry of bioactive compounds. 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